None of the references described or referred to herein are admitted to be prior art to the claimed invention.
Diagnostic assays are widely used in clinical diagnosis and health science research to detect or quantify the presence or amount of biological antigens, cell or genetic abnormalities, disease states, and disease-associated pathogens or genetic mutations in an organism or biological sample. Where a diagnostic assay permits quantification, practitioners may be better able to calculate the extent of infection or disease and to determine the state of a disease over time. Diagnostic assays are frequently focused on the detection of chemicals, proteins or polysaccharides antigens, nucleic acids, biopolymers, cells, or tissue of interest. A variety of assays may be employed to detect these diagnostic indicators.
Nucleic acid-based assays, in particular, generally include multiple steps leading to the detection or quantification of one or more target nucleic acid sequences in a sample. The targeted nucleic acid sequences are often specific to an identifiable group of cells, tissues, organisms, or viruses, where the group is defined by at least one shared sequence of nucleic acid that is common to members of the group and is specific to that group in the sample being assayed. A variety of nucleic acid-based detection methods are fully described by Kohne, U.S. Pat. No. 4,851,330, and Hogan, U.S. Pat. No. 5,541,308, the disclosures of each of which are hereby incorporated by reference.
A nucleic acid-based assay is performed, for example, in part by exposing a sample to a probe configured to exhibit specificity, under particular hybridization conditions, for a nucleic acid sequence belonging to the protein, cell, tissue, organism, or virus of interest. The sample is frequently treated in some manner to extract nucleic acids in a manner that they are eligible for hybridization.
Before or after exposing the target nucleic acid to a probe, the target nucleic acid can be immobilized by target-capture means, either directly or indirectly, using a “capture probe” bound to a substrate, such as a magnetic bead. Target capture probes are generally short nucleic acid sequences (i.e., oligonucleotide) capable of hybridizing with a sequence of nucleic acid that contains the target sequence. When magnetic beads comprise capture probes, magnets in close proximity to the reaction vessel are used to draw and hold the magnetic beads to the side of the vessel. Once the target nucleic acid is thus immobilized, the hybridized nucleic acid can be separated from non-hybridized nucleic acid present in the sample by, for example, aspirating fluid from the reaction vessel and optionally performing one or more wash steps.
In most instances, it is desirable to amplify the target sequence using any of several nucleic acid amplification procedures which are well known in the art. Methods of nucleic acid amplification are thoroughly described in the literature. PCR amplification, for instance, is described by Mullis et al. in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Methods in Enzymology, 155:335-350 (1987), the disclosure of each of which is hereby incorporated by reference. Examples of SDA can be found in Walker, PCR Methods and Applications, 3:25-30 (1993), Walker et al. in Nucleic Acids Res., 20:1691-1996 (1992) and Proc. Natl. Acad. Sci., 89:392-396 (1991). LCR is described in U.S. Pat. Nos. 5,427,930 and 5,686,272, the disclosure of each of which is hereby incorporated by reference. Examples of transcription-associated amplification (“TAA”) formats are provided, for example, in Burg et al. in U.S. Pat. No. 5,437,990; Kacian et al. in U.S. Pat. Nos. 5,399,491 and 5,554,516; and Gingeras et al. in International Application No. PCT/US87/01966 (published as International Publication No. WO 88/01302), and International Application No. PCT/US88/02108 (published as International Publication No. WO 88/10315), the disclosure of each of which is hereby incorporated by reference.
Detection of a targeted nucleic acid sequence frequently requires the use of a nucleic acid molecule having a nucleotide base sequence that is substantially complementary to at least a portion of the targeted sequence or its amplicon. Under selective assay conditions, the probe will hybridize to the targeted sequence or its amplicon in a manner permitting a practitioner to detect the presence of the targeted sequence in a sample. Techniques of effective probe preparation are known in the art. In general, however, effective probes are designed to prevent non-specific hybridization with itself or any nucleic acid molecule that will interfere with detecting the presence of the targeted sequence. Probes may include, for example, a label capable of detection, where the label is, for example, a radiolabel, a fluorophore or fluorescent dye, biotin, an enzyme, a chemiluminescent compound, or another type of detectable signal known in the art.
Because the probe hybridizes to the targeted sequence or its amplicon in a manner permitting detection of a signal indicating the presence of the targeted sequence in a sample, the strength of the signal is proportional to the amount of target sequence or its amplicon that is present. Accordingly, by periodically measuring, during the amplification process, a signal indicative of the presence of amplicon, the growth of amplicon overtime can be detected. Based on the data collected during this “real-time” monitoring of the amplification process, the amount of the target nucleic acid that was originally in the sample can be ascertained. Systems and methods for real time detection and for processing real time data to ascertain nucleic acid levels are described, for example, in Lair, et al., U.S. Pat. No. 7,932,081, “Signal Measuring System for Conducting Real-Time Amplification Assays,” the disclosure of which is hereby incorporated by reference.
To detect different nucleic acids of interest in a single assay, different probes configured to hybridize to different nucleic acids, each of which may provide detectibly different signals can be used. For example, different probes configured to hybridize to different targets can be formulated with fluorophores that fluoresce at a predetermined wavelength when exposed to excitation light of a prescribed excitation wavelength. Assays for detecting different target nucleic acids can be performed in parallel by alternately exposing the sample material to different excitation wavelengths and detecting the level of fluorescence at the wavelength of interest corresponding to the probe for each target nucleic acid during the real-time monitoring process. Parallel processing can be performed using different signal detecting devices constructed and arranged to periodically measure signal emissions during the amplification process, and with different signal detecting devices being configured to generate excitation signals of different wavelengths and to measure emission signals of different wavelengths. Suitable signal detecting devices include fluorometers, such as the fluorometer described below.
Thermal melt analysis, or melting curve analysis, encompasses an assessment of the dissociation-characteristics of double-stranded DNA during heating to identify specific genotypes within a target nucleic acid. The information gathered can be used to infer the presence and identity of single-nucleotide polymorphisms. More specifically, the energy required to break the base-base hydrogen bonding between two strands of DNA is dependent on their length, GC-content (or guanine-cytosine content), and their complementarity. By heating a reaction-mixture that contains double-stranded DNA sequences and measuring dissociation against temperature, a variety of attributes can be inferred. Originally strand dissociation was observed using UV absorbance measurements, but techniques based on fluorescence measurements are now the most common approach. The temperature-dependent dissociation between two DNA-strands can be measured using a DNA-intercalating fluorophore, such as SYBR green, EvaGreen or fluorophore-labeled DNA probes. In the case of SYBR green, the dissociation of the DNA during heating is measurable by the large reduction in fluorescence that results. Alternatively, juxtapositioned probes, one featuring a fluorophore and the other featuring a suitable quencher can be used to determine the complementarity of the probe to the target sequence. For example, though a variety of other methods are known in the art, a graph of the negative first derivative of the melting-curve may make it easier to pin-point the temperature of dissociation (defined as 50% dissociation), by virtue of the peaks thus defined.
Melt curve analysis describes a method where the temperature dependent dissociation of two strands of nucleic acids is measured. To perform the melt curve analysis, the temperature of a sample, and/or an amplicon contained therein, is raised while monitoring a signal emitted by the sample, such as the fluorescence of a fluorophore labeled probe. As the temperature rises, the dissociation of the probe and the amplicon can be measured as detectable change in the signal, such as by a decrease in fluorescence. A melt station holds one or more receptacles containing sample materials, e.g., amplicon, and subjects the contents of the receptacles to thermal energy to raise the temperature of the amplicon along a controlled temperature profile while monitoring signal, e.g., fluorescence, emitted by the contents. Where the detected signal is fluorescence, the fluorescence may be monitored in one or more wavelengths. The procedure results in a melt curve of fluorescence vs. time. Differences in the melt temperature can be used to discriminate variations in the sequence of the amplicon. For example, the mutant and wild type strands may exhibit markedly different melt temperatures.
Typically, thermal melt analysis is performed on molecular diagnostic instruments that process samples in batch. A group of samples, i.e., the “batch,” is placed in the instrument—typically a thermal block, and the instrument is operated to perform the thermal melt and the thermal melt analysis on all samples substantially simultaneously. Instrument operation continues until the assay has been completed for all samples placed in the instrument. After completion of the assay, the operation of the instrument is stopped, or paused, the batch of samples is removed, the temperature of the instrument, or thermal block, is ramped down to a particular starting temperature, and then a subsequent batch of samples may be placed in the instrument and the process repeated.
Typically melt analysis is performed by placing a receptacle holding a reaction liquid into an instrument which ramps the temperature of the reaction liquid up by ramping the temperature of a component, often referred to as a thermal, block, of the instrument. The temperature of the block is ramped, according to a pre-defined temperature profile, slowly enough so that the temperature of the reaction liquid accurately follows the temperature of the block. The temperature of the block can be changed slowly and linearly, or it can be changed in stepwise fashion while holding the temperature of each step long enough for the reaction liquid to reach steady state at each temperature step. The temperature of the block must start at or below the lowest analysis temperature and end at or above the highest analysis temperature to ensure that the temperature of the reaction liquid is known throughout the melting process. To get ready for the next receptacle, i.e., batch, the temperature of the block must ramp down to the start temperature. The total throughput of the instrument is limited by the speed at which the temperature of the reaction liquid can be changed from the start temperature to the end temperature and the speed at which the temperature of the block can be returned back to the start temperature to get ready for the next batch. The analysis time is also limited by the speed at which the temperature of the reaction liquid within the receptacle can follow the block temperature.
Other diagnostic instruments process samples in a serial (also known as a linear or pipeline) manner, as opposed to a batch manner. Samples are sequentially and continuously processed through the instrument, with different steps of the process being performed on different samples in a parallel manner. One sample may be completing the assay process, while another is just beginning the process. Thus, processing on all the samples is not started or completed at the same time, and assays may be completed on a periodic basis, for example, once every five minutes, during operation of the instrument.
For automated instruments that process samples in a serial fashion, in order to maintain an exemplary sequence of completing one sample assay every five minutes, or other desired interval, the melt station must be able to process one reaction receptacle at a time and complete the thermal melt cycle in preferably one five-minute interval. If the thermal melt station is not able to maintain the desired frequency by completing the thermal melt cycle within the specified time interval, it becomes necessary to employ two, or more, thermal melt stations operating in parallel. The need to slowly ramp the temperature from the starting temperature to the ending temperature and then back to the starting temperature, as described above, as well as the time lag that may be required for the contents of the receptacle to reach thermal equilibrium with the thermal block creates a challenge to design a thermal melt station that can complete the thermal melt procedure within the time interval needed to maintain the desired through put of a serial-processing molecular diagnostic instrument.